Summary

TRAIL-R2 is one of the TRAIL death receptors, and its agonists have been subject to many cancer treatment clinical trials to date. The most important obstacle to cancer treatment with TRAIL is the resistance, which may be intrinsic or may develop due to long-term exposure. One of the most prominent causes of TRAIL resistance is TRAIL-R2 endocytosis and its migration to the nucleus. In order to both understand the causes of TRAIL-R2 endocytosis and uncover the factors affecting TRAIL sensitivity, -and thus pave the way for TRAIL therapy of cancer, TRAIL-R2 signal transduction, especially the post-translational modifications must be fully understood. In our bioinformatic analyzes, we detected a tyrosine phosphorylation motif(YxxM) on the death domain (DD) of TRAIL-R2, which was conserved among species. When we searched Phosphosite database regarding this phosphorylation, which has never been reported in the literature, we noticed that phosphorylation of this motif was detected in the mass-spectrometry analysis results of MKN-45 and K562 cell lines. To determine whether we could demonstrate this phosphorylation that has never been tested in a wet lab environment, as a preliminary study, we examined the levels of TRAIL-R2 tyrosine phosphorylation in the TRAIL-resistant human cervical cancer cell line HeLa with and without TRAIL treatment. We showed that even in the absence of ligand, TRAIL-R2 is phosphorylated, level of which reached its maximum with 15 minutes TRAIL treatment. Since TRAIL-R2 tyrosine phosphorylation motif is also fully compatible with the clathrin-dependent endocytosis motif(YXXØ), it is highly likely that this phosphorylation plays a role in TRAIL resistance by regulating TRAIL-R2 endocytosis and intracellular trafficking. Also, since this phosphorylation motif is located on DD, it may play a role in regulating the physical interaction of TRAIL-R2 with other TRAIL receptors and signal transduction partners. In our project proposal, we first aim, following demonstration of TRAIL-R2 tyrosine phosphorylation in a wet lab setting, to identify the responsible tyrosine kinase.  We then aim to obtain HeLa cell lines that will express Y>D mutant which will mimic TRAIL-R2 phosphorylation and Y>F mutant which will inhibit phosphorylation. Next, we will examine how TRAIL-R2 tyrosine phosphorylation affects TRAIL-R2 expression in different cellular compartments in these cell lines. Surface expression will be analysed by flow cytometry and expression in lipid rafts, nuclei, and cytosol will be investigated by western blot (WB). We will determine how the interactions of wild-type and mutant TRAIL-R2 with their binding partners are affected by immunoprecipitation(IP) followed by WB, the impact on proliferative pathways by WB, the cell survival by WST, and the proliferation by BrdU uptake test. In order to reveal the effects of TRAIL-R2 tyrosine phosphorylation on metastasis, we will perform in vitro wound healing test, and will transplant into NOD-SCID mice to examine the effect of TRAIL-R2 tyrosine phosphorylation on tumor engraftment. In our project, which will be conducted under the consultancy of Prof Dr Ahter Şanlıoğlu, a specialist in TRAIL and gene therapy, the production of adeno-associated viral vectors (AAV) will be carried out by Dr. Pelin Dilsiz Erim, who has an intensive experience in this field. Work packages related to intracellular localization of TRAIL-R2 will be carried out by Dr Ufuk Mert, whose doctoral thesis was in this field. The project coordinator has completed her doctoral thesis on TNF receptor-1 (TNFR1) phosphorylations and has experience with all the experiments to be carried out in the Project. She will transfer her experience to a masters and a doctoral student. In addition, because a hitherto unknown aspect in TRAIL/TRAIL-R2 signal transmission will be revealed as a result of our project, we hope that our results can both be presented at international congresses and be published in a high-impact factor journal.